糖化中麦芽淀粉酶系热稳定性的研究

本文研究了糖化过程中麦芽淀粉酶系的热稳定性、不同品种麦芽间淀粉酶热稳定性的差异及对麦汁糖组分的影响。结果表明糖化中45～65℃α-淀粉酶活力变化不显著;β-淀粉酶在60℃左右时酶活最大;极限糊精酶在45～60℃时酶活力稳定;温度超过60℃,β-淀粉酶和极限糊精酶的酶活显著下降,温度超过65℃α-淀粉酶活力下降明显。不同品种麦芽中淀粉酶系的热稳定性存在差异,其中β-淀粉酶热稳定性的差异最为显著。麦芽品种对麦汁可发酵性的影响明显。研究表明β-淀粉酶活力及其热稳定性是决定麦汁可发酵性的主要因素。     

蒙脱石固定化α-淀粉酶和糖化酶的研究

以蒙脱石为载体,利用吸附法分别固定α-淀粉酶、糖化酶以及共固定化α-淀粉酶和糖化酶。α-淀粉酶和糖化酶最佳固定化温度分别是20℃和30℃,最佳固定化pH是6.5和4.5。共固定化最佳条件为淀粉酶(U):糖化酶(U):蒙脱石(g)为15:7.5:0.2,pH为5.5;温度为20℃。固定化α-淀粉酶、固定化糖化酶和共固定化酶的最适pH分别为6.0、4.5和5.5,最适反应温度分别是60、60℃和55℃。蒙脱石固定化α-淀粉酶、固定化糖化酶和共固定化酶的稳定性均较好,尤其是共固定化酶表现突出。     

氨基化硅胶作载体固定化α-淀粉酶的研究

采用5 种硅胶作为载体,戊二醛作偶联剂,制备了固定化α- 淀粉酶。对制备固定化α- 淀粉酶的硅胶目数、偶联剂浓度、温度、pH 值的影响因素进行了研究和优化。并对固定化酶和游离酶的酶学性质进行了比较。结果表明：固定化酶的最适温度为75℃,比游离酶提高了10℃,固定化酶的热稳定性优于游离酶。与游离酶相比,其对酸碱的适应性、贮存稳定性、操作稳定性也都有较明显改善。固定化酶的Km 值略低于游离酶,前者为2.13mg/ml,后者为1.02mg/ml。     

水牛乳过氧化物酶特性的研究

以第三代摩拉×本地奶水牛的新鲜乳为原料,对乳中过氧化物酶的最适温度、热稳定性、最适pH值、pH值稳定性以及光稳定性用ABTS进行测定。结果表明,乳中过氧化酶最适合温度在55℃附近,且在40℃以下稳定性较好;最适pH值在5.5附近,pH值为3.0～8.0都很稳定;日光灯照射条件下,酶的稳定性较差而避光条件下相对较好。这些研究将为南方以及广西地区水牛乳的保鲜提供了部分依据。     

Thermobifida fusca麦芽三糖淀粉酶的重组表达及其在麦芽三糖制备中的应用

麦芽三糖淀粉酶以淀粉或麦芽糊精为原料,可生产麦芽三糖,在食品、饮料等行业具有广泛的应用前景。实验将Thermobifida fusca NTU22来源的麦芽三糖淀粉酶在枯草芽孢杆菌WS11中进行重组表达,并进行酶学性质分析,重组酶的最适温度和pH分别是55℃和6.0。在此基础上探究了重组酶作用于麦芽糊精生产麦芽三糖的最佳条件。结果表明,以15%浓度的麦芽糊精(DE 5-7)作为底物时,酶转化的最佳条件是温度55℃、pH 5.5、加酶量60 U/g(底物)、普鲁兰酶加酶量32 U/g(底物)、反应时间11 h,此时得到麦芽三糖转化率44.4%。该研究为工业制备麦芽三糖提供了理论依据。     

工业酿酒酵母重组β-1,3-1,4-葡聚糖酶部分酶学性质的研究

用刚果红法测定β-1,3-1,4-葡聚糖酶的酶活力,研究重组酿酒酵母(S.cerevisiae)菌株SC-βG分泌表达的重组β-1,3-1,4-葡聚糖酶的部分酶学性质,并与出发菌株枯草芽孢杆菌(B.subtilis)表达的原始酶的性质进行比较。结果表明,重组酶保持了与原始酶相同的底物专一性。 重组酶的最适反应温度为35℃,而原始酶为55℃。重组酶的热稳定性也发生了改变,40℃热处理20min只保留63.4%的最初酶活力,但温度再升高时对热处理敏感度降低,70℃的热处理20min仍保留45.9%的最初酶活力;而原始酶50℃时稳定,60℃以上的热处理酶活力损失很大。与原始酶相比,重组酶的最适pH值下降为pH5.0,而原始酶为pH6.5;相比原始酶在pH7.0有最大稳定性,重组酶在pH5.5时有最大稳定性。重组β-1,3-1,4-葡聚糖酶的最适反应条件与原始酶相比更接近啤酒的实际生产条件。     

双酶协同酶解木薯淀粉的研究

本文研究了α-淀粉酶(酶活:4,000 U/g,最适pH 5.5～6.5,最适温度50～55℃)和糖化酶(酶活:100,000 U/g,最适pH 4.0～4.5,最适温度58～62℃)协同水解制备木薯微孔淀粉的工艺条件.结果表明木薯淀粉的水解率为50%时质量最好,此时的工艺条件为:加酶量(α-淀粉酶与糖化酶的质量比为3:1)为干基淀粉质量的0.50%,反应时间12 h,反应温度55℃,反应pH 5.0.     

关于添加金属离子对国产大麦芽酶系酶活力影响的研究

国产大麦芽的酶活力不如进口大麦芽的酶活力强,其中原因之一是国产大麦中某些金属离子含量低,而这些离子对大麦中的某些酶有抑制和激活作用。实验发现:当添加Na+、K+、Mg2+、Zn2+和Cu2+浓度分别达到80×10-6、60×10-6、40×10-6、20×10-6、20×10-6时,制麦酶系中的α-淀粉酶、β-淀粉酶、蛋白酶和纤维素酶活力都达到最大值,提高幅度明显。故可通过添加某些金属离子来强化制麦酶系的酶活力,提高其生产性能。     

曲霉型豆豉酿造中淀粉酶作用条件研究

研究曲霉型豆豉酿造用米曲霉沪酿3.042产淀粉酶的最适作用条件。利用大豆豆汁培养基对米曲霉沪酿3.042菌株进行培养,并用(NH4)2SO4溶液对所产淀粉酶进行沉淀、脱盐处理后,研究该酶的最适作用pH值及pH稳定性、最适作用温度及热稳定性以及NaCl浓度对酶活力的影响。结果显示:该淀粉酶最适作用温度为55℃,最适作用pH值为4.5,酶活力为18.0mg/h,在30~50℃,pH 4.0~6.0范围内其酶活力较稳定;NaCl对淀粉酶酶促反应有明显的抑制作用,随着NaCl浓度的升高,其抑制效果越明显。     

玉米萌发过程中淀粉酶性质的研究

本实验研究了玉米萌发过程中各种因素对其中所含淀粉酶活力的影响,并研究了最佳萌发条件下,总淀粉酶、α-淀粉酶和β-淀粉酶活力的变化情况,通过SDS-PAGE凝胶电泳确定了α-淀粉酶和β-淀粉酶的分子量,以SephadexG-100色谱对两种酶进行了分离。结果表明,水分含量、温度、光照以及赤霉素等因素对玉米淀粉酶活力均有影响,在水分35%、温度30℃、赤霉素30mg/L和自然光照条件下萌发,玉米的淀粉酶活力最高;在萌发过程中,α-淀粉酶活力增加得比较缓慢,而且活力也大大低于β-淀粉酶,总淀粉酶和β-淀粉酶活力上升都比较迅速;β-淀粉酶的分子量为189.82×103D,α-淀粉酶的分子量为54.26×103D。     

A New Approach to Limit Dextrinase and its Role in Mashing*

Limit dextrinase (EC 3.1.2.41) is a debranching enzyme catalyzing the hydrolysis of α-1,6-glucosidic linkages in starch. The role of this debranching enzyme in beer brewing has been questioned due to its assumed heat lability. In the present work the effectiveness of limit dextrinase was studied under conditions mimicking brewery practice rather than in a buffer solution. It was demonstrated that typical conversion temperatures of 63–65 °C and a mash pH of 5.4–5.5 favour the action of malt limit dextrinase. The temperature optimum for the limit dextrinase of a malt extract was 60–62.5 °C, as opposed to 50 °C for purified limit dextrinase. Lowering the mash pH from 5.8 to 5.4 increased wort fermentability due to increased limit dextrinase activity. Wort fermentability was more strongly correlated to the free limit dextrinase activity of malt than to the α- and β-amylase activities.     

The Advantages of Using Natural Substrate‐Based Methods in Assessing the Roles and Synergistic and Competitive Interactions of Barley Malt Starch‐Degrading Enzymes

Existing methods of assay of malt starch‐degrading enzymes were critically appraised. New methods based on natural substrates, namely starch and its natural intermediate‐derivative, were developed for all the enzymes, except limit dextrinase for which pullulan was used. Thermostability, optimal temperatures and pHs were established. α‐Amylase and limit dextrinase were the most thermostable and β‐amylase, α‐glucosidase and maltase were the least stable while diastase occupied an intermediate position. The optimal temperatures were congruent with thermostability, β‐ amylase having the lowest (50°C) and α‐amylase the highest (65°C) with the remaining enzymes, including diastase, falling in between. In contrast, α‐amylase has the lowest optimal pH (pH 4.5) and β amylase the highest (pH 5.5) while the others have pHs in between the two values. The roles of the enzymes were evaluated taking into account the level of activity, thermostability, optimum pH, the nature of the product(s), and the relevance to brewing. β‐Amylase production of maltose was synergistically enhanced, mostly by α‐amylase but also limit dextrinase. α‐Glucosidase and maltase are unimportant for brewing, because of their low activity and the negative impact on β‐amylase activity and the negative effect of glucose on maltose uptake by yeast. The starch‐degrading enzymes (diastase) in a gram of malt were able to degrade more than 8 g boiled starch into reducing sugars in 10 min at 65°C. The latter, suggests that it will be possible to gelatinise most of the malt starch at a higher temperature and ensure its hydrolysis to fermentable sugars by mixing with smaller portions of malt and mashing at lower temperatures e.g. 50–60°C.     

Limit Dextrinase — Does Its Malt Activity Relate to Its Activity During Brewing?

Limit dextrinase (EC 3.2.1.142) hydrolyses α‐1,6 glucosidic bonds in amylopectin and branched dextrins. Measurement of limit dextrinase activity during fermentation of unboiled wort at the pH of the wort has shown that its activity increases almost 10 fold during the first 10–15 h of fermentation and this increase in activity is unaffected by the presence of leupeptin, a cysteine protease inhibitor. The increase in activity seen when assays were carried out at pH 5.5 was much smaller and was reduced by leupeptin. The activity of limit dextrinase declined slowly during the latter part of the fermentation. It was established that the optimum pH for rapid extraction and assay of malt limit dextrinase in the absence of a reducing agent is approximately 4.5, but in the presence of dithiothreitol, at pH 5.5, activities 2–3 times higher can be obtained after 5 h extraction (600–700 mU/g dry weight). Limit dextrinase activities after 1 h extraction at mashing temperatures were below 20 mU/g dry weight if the mash pH was below 5.0. It is concluded that at pHs below 5.0, where limit dextrinase activity is below its optimum for activity, limit dextrinase activity increases due to dissociation of the inhibitor/enzyme complex. The protection from mashing temperatures of 65°C afforded by the inhibitor is lost at these lower pHs.     

小麦芽脂肪酶酶学性质以及制麦过程的酶活变化

以对硝基苯酚丁酸酯为底物,确定了小麦芽脂肪酶的酶学性质:最适温度为37℃;最适pH为8.0;4～35℃脂肪酶的热稳定性较好,35℃下保温60 min后其活力仍可保持在88.37%,45℃保温60 min后其活力保持在46.81%,55℃保温60 min酶失活;pH5.5～6.0范围内对脂肪酶的破坏力相对较小,保温60 min其活力仍可分别保持在95.11%和91.11%;Fe3+对脂肪酶有较强的抑制作用;Cu2+、Mn2+、Al3+对脂肪酶的抑制作用较小;Ca2+、EDTA对脂肪酶有较强的激活作用。小麦制麦过程中发芽第4天酶活达到最大值,为8.52 u/g,随后酶活下降;干燥会破坏部分脂肪酶的酶活,损失率为27.06%。小麦芽籽粒、胚芽、胚根中的脂肪酶酶活依次降低,分别为8.01 u/g、7.55 u/g、6.83 u/g;协定法麦汁糖化过程中,无胚芽麦芽脂肪酶的酶活比有胚芽的低,且酶的失活速度也比较大,在55 min(70℃)时,脂肪酶完全失活。     

FACTORS INFLUENCING BETA-AMYLASE ACTIVITY IN SORGHUM MALT

An investigation into factors influencing beta-amylase activity in sorghum malt confirmed that ungerminated sorghum grain exhibited essentially no beta-amylase activity. Malted sorghum had beta-amylase activity less than 25% of the level in barley malt. Neither reducing agents nor papain affected beta-amylase activity in sorghum, indicating that the enzyme is not in a bound form, unlike in barley. Isoelectric focusing indicated that sorghum beta-amylase comprises just one major and one minor isozyme of pl approximately 4.4–4.5, unlike the many isozymes all of higher pl in barley. However, like barley, sorghum beta-amylase was more temperature-labile than its alpha-amylase. Beta-amylase activity in sorghum malt was increased by germination time, high germination moisture and over the germination temperature range investigated (24–32°C), 24°C gave the highest activity. The beta-amylase activity of sorghum malts was significantly correlated with malt diastatic power, despite the fact that alpha-amylase and not beta-amylase is the predominant diastatic enzyme in sorghum malt.     

两步水解法制备玉米-大豆复合抗氧化肽及其稳定性研究

采用麦芽粉和碱性蛋白酶Alcalase两步水解玉米醇溶蛋白和大豆分离蛋白的混合物(质量比1∶1)制备复合抗氧化肽,以水解度、可溶性蛋白含量和抗氧化活性为指标,对第二步Alcalase的水解条件进行优化,确定两步水解法制备复合抗氧化肽的最适条件。另外,对复合抗氧化肽相对分子质量分布、稳定性和氨基酸组成进行了研究。结果表明,制备复合抗氧化肽的最适条件为在底物质量分数10%、pH 5.5、50℃、酶(麦芽粉)底质量比30%条件下水解3 h后,再在底物质量分数10%、pH 8.5、60℃、酶(Alcalase)底质量比0.75%条件下继续水解3 h。在最适条件下,水解度、可溶性蛋白含量、羟自由基清除率和亚铁离子螯合率分别为36.32%、36.83 mg/mL、40.01%和86.83%,相对分子质量主要分布在300~6 500 Da (含量92.44%)范围内,必需氨基酸指数(EAAI)值为8.02(n=9),属于优质蛋白源。复合抗氧化肽具有热稳定性,在中性及碱性条件下稳定,但不耐受酸性(pH 3~5)、4℃贮存和反复冻融的加工条件。     

4种常用蛋白酶对牛骨蛋白的酶解动力学研究

对碱性蛋白酶、中性蛋白酶、胃蛋白酶、胰蛋白酶水解牛骨蛋白的动力学特征进行较为系统的研究。以酶解初速度为评价指标,得到了4 种酶的最佳反应温度与pH 值,其中碱性蛋白酶最佳温度为55℃,pH11.7;中性蛋白酶最佳温度为45℃,pH7.3;胃蛋白酶最佳温度为30℃,pH1.5;胰蛋白酶最佳温度为55℃,pH9.3。4种蛋白酶按Km 值排序从小到大依次是：胃蛋白酶＜胰蛋白酶＜碱性蛋白酶＜中性蛋白酶;Vmax 从大到小依次是：胃蛋白酶＞碱性蛋白酶＞胰蛋白酶＞中性蛋白酶。     

大麦芽极限糊精酶的分离纯化及酶学特性分析

将大麦芽提取的极限糊精酶粗酶液,利用硫酸铵沉淀、离子交换层析和凝胶过滤色谱等分离方法对极限糊精酶进行逐步分离纯化。结果表明：纯化倍数为31.23,回收率为8.81%。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后,图谱显示样品具有较高的纯度,分子质量约为97kD。同时研究了纯化前后极限糊精酶在不同作用环境下酶的活性变化,发现纯化后样品在温度45℃和pH 5.5左右具有最大酶活性,与粗酶液中酶活性相比具有明显差异。在体系中添加不同浓度的金属离子,结果发现,Mg2+、Ca2+、Mn2+在浓度较低时,对酶活性具有激活作用,而浓度较高时,则具有抑制作用;整体上,K+对酶活性影响不大;Zn2+、Fe2+对酶活性具有抑制作用。     

超声波辅助酶法水解鱼鳞胶原蛋白的工艺研究

采用超声波辅助木瓜蛋白酶水解鱼鳞胶原蛋白。研究超声波功率和作用时间对酶解反应的影响,应用正交设计优化酶解工艺条件。结果表明,最佳酶解工艺条件为:酶用量4 g/L、温度55℃、pH 5.5,在超声波功率为500 W、作用20 min后,继续酶解5 h。在此条件下,鱼鳞胶原蛋白的水解度为25.6%。与常规酶解方法比较,常规酶解8 h水解度为16.6%。说明超声波对木瓜蛋白酶水解鱼鳞胶原蛋白有促进作用。     

聚乙烯醇环氧化载体制备及在植酸酶固定化中的应用

该文以聚乙烯醇为原料,利用戊二醛为交联剂,聚合得到交联球形聚乙烯醇,并将其与环氧氯丙烷反应用以固载环氧基团,得到可用于酶固定化的载体。讨论了交联聚乙烯醇球环氧基的固载条件和酶的固定化条件。植酸酶经过固定化后,机械性能和化学稳定性都得到提高,可以重复多次对植酸钠进行水解反应。同时对该自由酶和固定化酶进行酶学特性研究。发现自由酶和固定化酶的最适pH值分别为5.5和7.0,最适反应温度分别为55℃和70℃。植酸酶经固定化,提高了酶的操作、温度、pH值和贮藏稳定性。